As molecular biology has helped to define regions of proteins that contribute to a particular biological activity, it has become desirable to synthesize short peptides to mimic or inhibit those activities. Many of the disadvantages encountered in therapeutic, diagnostic and industrial settings with purified proteins or proteins produced by recombinant means could easily be avoided by short synthetic peptides. For instance, synthetic peptides offer advantages of specificity, convenience of sample or bulk preparation, lower relative cost, high degree of purity, and long shelf-life.
Despite the great promise of synthetic peptides, precise sequence and binding data are not available for most proteins of significant medical, veterinary, agricultural or industrial interest. Even when the sequence of a protein is known, the process of identifying short sequences which are responsible for or contribute to a biological activity may be extremely tedious, if not impossible in many instances.
Thus, the ability to efficiently screen very large peptide libraries for desired binding activities would be of enormous interest. It would enable the identification of novel agonists and antagonists for receptors, the isolation of specific inhibitors of enzymes, provide probes for structural and functional analyses of binding sites of many proteins, and ligands for many other compounds employed in a wide variety of applications.
Recent advances in peptide chemistry and molecular biology have resulted in the development of methods for preparing and evaluating extremely large peptide libraries.
The generation of large numbers of peptide sequences by the cloning and expression of randomly-generated mixtures of oligonucleotides is possible in the appropriate recombinant vectors. See, e.g., Oliphant et al., Gene 44:177-183 (1986). Such a large number of compounds can be produced, however, that methods for efficient physical and genetic selection are required. Without such methods the usefulness of these large peptide libraries in providing ligands of potential interest may be lost.
Large numbers of randomly or specifically directed peptides have been synthesized and assayed for activity, usually binding to an antibody to determine the residues which comprise the epitope recognized by the antibody. The strategies employed may be divided into two categories. In the first a mixture of peptides is exposed to a receptor and the resultant binding is used to separate the active peptides from the inactive peptides. The identity of the active peptides is then determined by the techniques of molecular biology, as described in, for example, Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990), Devlin et al., Science 249:404-406 (1990) or Scott and Smith, Science 249: 386-390 (1990). Problems associated with this approach involve the considerable time and effort in working with large numbers of transformed organisms. Alternatively, the peptide sequence may be determined by means of peptide chemistry, as generally described in Lam et al., Nature 354:82-84 (1991), although in this approach the peptides must be bound to a polymer resin and thus may be limited or unavailable to interact with some receptors. Moreover, the method of Lam et al. requires that a visual label be attached to the receptor of interest, which may itself pose problems.
In the second strategy the synthesis of the peptide analogs is compartmentalized and this knowledge is used to determine the identity of the active peptides. See generally, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al., in Synthetic Peptides as Antigens, Ciba Foundations Symposium, 119, Porter and Wheelan, eds., pp 131-149, Wiley, N.Y. (1986); Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985); Houghten et al., Nature 354:84-86 (1991); and Fodor et al., Science 251:767-773 (1991). The method of Geysen et al. generally suffer from the same disadvantages as the method of Lam et al., in that the peptide must be anchored to a solid support. The method of Fodor et al. also involves anchoring the peptide to a solid support, and further involves difficult chemistry, and thus may not be feasible from a practical standpoint. Houghten et al. describes an iterative strategy, but requires special equipment and expertise and thus may be of limited widespread application.
What are needed in the art are methods which avoid the problems associated with large numbers of transformed organisms as well as the limitations of methods which employ the compartmentalized synthesis of peptide analogs, as discussed above, and which may be completed more rapidly than currently available procedures. The present invention fulfills these and other related needs. Contrary to previously disclosed methods, the present invention describes methods wherein mixtures of peptides are synthesized and evaluated, and subsequent iterative variations in the mixtures allow a determination of the active peptide or peptides. Therefore, the present methodology provides for efficient screening and selection from large peptide libraries, and further provides substantial time and monetary savings in the identification and isolation of the novel peptides.